ObjeCtivesTo provide an early risk assessment of extending screening intervals beyond five years for a human papillomavirus (HPV) based cervical screening programme in the Netherlands. Design 14 year follow-up of a population based randomised cohort from the POBASCAM randomised trial.setting Organised cervical screening in the Netherlands, based on a programme of three screening rounds (each round done every five years).PartiCiPants 43 339 women aged 29-61 years with a negative HPV and/or negative cytology test participating in the POBASCAM trial.interventiOns Women randomly assigned to HPV and cytology co-testing (intervention) or cytology testing only (control), and managed accordingly. Main OutCOMe MeasuresCumulative incidence of cervical cancer and cervical intraepithelial neoplasia (CIN) grade 3 or worse (CIN3+). Associations with age were expressed as incidence rate ratios. In HPV positive women, reductions in CIN3+ incidence after negative cytology, HPV type 16/18 genotyping, and/or repeat cytology were estimated. resultsThe cumulative incidence of cervical cancer (0.09%) and CIN3+ (0.56%) among HPV negative women in the intervention group after three rounds of screening were similar to the cumulative among women with negative cytology in the control group after two rounds (0.09% and 0.69%, respectively). Cervical cancer and CIN3+ risk ratios were 0.97 (95% confidence interval 0.41 to 2.31, P=0.95) and 0.82 (0.62 to 1.09, P=0.17), respectively. CIN3+ incidence was 72.2% (95% confidence interval 61.6% to 79.9%, P<0.001) lower among HPV negative women aged at least 40 years than among younger women. No significant association between cervical cancer incidence and age could be demonstrated. CIN3+ incidence among HPV positive women with negative cytology, HPV 16/18 genotyping, and/or repeat cytology was 10.4 (95% confidence interval 5.9 to 18.4) times higher than among HPV negative women. COnClusiOnsLong term incidences of cervical cancer and CIN3+ were low among HPV negative women in this study cohort, and supports an extension of the cervical screening interval beyond five years for women aged 40 years and older. HPV positive women with subsequent negative cytology, HPV16/18 genotyping, and/or repeat cytology have at least a fivefold higher risk of CIN3+ than HPV negative women, indicating that HPV based programmes with long intervals (>five years) should be implemented with risk stratification.trial registratiOn POBASCAM trial number ISRCTN20781131. IntroductionRandomised controlled clinical trials have shown that screening for high risk human papillomavirus (HPV) leads to earlier detection of cervical intraepithelial neoplasia (CIN) grade 3 or worse (CIN3+) than cytology, [1][2][3][4] and provides better protection against cervical cancer. 5-10 Primary HPV testing shows positive results more often than primary cytology testing in the general screening population, and subsequent triage testing of HPV positive women (by use of cytology and of genotyping of HPV subtypes 16 and 18) has been recommend...

Aims Gene promoter hypermethylation is recognized as an essential early step in carcinogenesis, indicating important application areas for DNA methylation analysis in early cancer detection. The current study was set out to assess the performance of CADM1, MAL and miR124-2 methylation analysis in cervical scrapes for detection of cervical and endometrial cancer.Methods A series of cervical scrapes of women with cervical (n=79) or endometrial (n=21) cancer, CIN3 (n=16) or CIN2 (n= 32), and women without evidence of CIN2 or worse (n=120), was assessed for methylation of CADM1, MAL and miR124-2. Methylation analysis was done by the PreCursor-M assay, a multiplex quantitative methylation-specific PCR.Results All samples of women with cervical cancer (79/79, 100%), independent of the histotype, and 76% (16/21; 95% CI:58.0-94.4) of women with endometrial cancer scored positive for DNA methylation of at least one of the three genes. In women without cancer, methylation frequencies increased significantly to severity of disease, from 19.2% (23/120; 95% CI:12.1-26.2) in women without CIN2 or worse to 37.5% (12/32; 95% CI: 20.7-54.3) and 68.8% (11/16; 95% CI:46.0-91.5) in women with CIN2 and CIN3, respectively. Not only overall methylation positivity, but also the number of methylated genes increased proportionally to the lesion severity.Conclusions DNA methylation analysis of CADM1, MAL and miR124-2 in cervical scrapes consistently detects cervical cancer and the majority of CIN3 lesions, and has the capacity to broaden its use on cervical scrapes through the detection of a substantial subset of endometrial carcinomas.

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AimsTo investigate the accuracy and reproducibility of a scoring system for cervical intraepithelial neoplasia (CIN1–3) based on immunohistochemical (IHC) biomarkers Ki-67 and p16ink4a.Methods115 cervical tissue specimens were reviewed by three expert gynaecopathologists and graded according to three strategies: (1) CIN grade based on H&E staining only; (2) immunoscore based on the cumulative score of Ki-67 and p16ink4a only (0–6); and (3) CIN grade based on H&E supported by non-objectified IHC 2 weeks after scoring 1 and 2. The majority consensus diagnosis of the CIN grade based on H&E supported by IHC was used as the Reference Standard. The proportion of test positives (accuracy) and the absolute agreements across pathologists (reproducibility) of the three grading strategies within each Reference Standard category were calculated.ResultsWe found that immunoscoring with positivity definition 6 yielded the highest proportion of test positives for Reference Standard CIN3 (95.5%), in combination with the lowest proportion of test positives in samples with CIN1 (1.8%). The proportion of test positives for CIN3 was significantly lower for sole H&E staining (81.8%) or combined H&E and IHC grading (84.8%) with positivity definition ≥CIN3. Immunoscore 6 also yielded high absolute agreements for CIN3 and CIN1, but the absolute agreement was low for CIN2.ConclusionsThe higher accuracy and reproducibility of the immunoscore opens the possibility of a more standardised and reproducible definition of CIN grade than conventional pathology practice, allowing a more accurate comparison of CIN-based management strategies and evaluation of new biomarkers to improve the understanding of progression of precancer from human papillomavirus infection to cancer.

IntroductionTo evaluate the performance of hypermethylation analysis of ASCL1,LHX8 and ST6GALNAC5 in physician‐taken cervical scrapes for detection of cervical cancer and cervical intraepithelial neoplasia (CIN) grade 3 in women living with HIV (WLHIV) in South Africa.MethodsSamples from a prospective observational cohort study were used for these analyses. Two cohorts were included: a cohort of WLHIV who were invited for cervical screening (n = 321) and a gynaecologic outpatient cohort of women referred for evaluation of abnormal cytology or biopsy proven cervical cancer (n = 108, 60% HIV seropositive). Cervical scrapes collected from all subjects were analysed for hypermethylation of ASCL1,LHX8 and ST6GALNAC5 by multiplex quantitative methylation specific PCR (qMSP). Histology endpoints were available for all study subjects.ResultsHypermethylation levels of ASCL1,LHX8 and ST6GALNAC5 increased with severity of cervical disease. The performance for detection of CIN3 or worse (CIN3+) as assessed by the area under the receiver operating characteristic (ROC) curves (AUC) was good for ASCL1 and LHX8 (AUC 0.79 and 0.81 respectively), and moderate for ST6GALNAC5 (AUC 0.71). At a threshold corresponding to 75% specificity, CIN3+ sensitivity was 72.1% for ASCL1 and 73.8% for LHX8 and all samples from women with cervical cancer scored positive for these two markers.ConclusionsHypermethylation analysis of ASCL1 or LHX8 in cervical scrape material of WLHIV detects all cervical carcinomas with an acceptable sensitivity and good specificity for CIN3+, warranting further exploration of these methylation markers as a stand‐alone test for cervical screening in low‐resource settings.

In this HIV-infected South African population, stratifying hrHPV-positive women with reflex methylation analysis detects all cervical carcinomas and yields an acceptable sensitivity and specificity for CIN3+.

Post-treatment monitoring by CADM1/MAL-methylation analysis identifies women with an increased risk of rCIN2/3. Our results confirm previous data indicating that CADM1/MAL-methylation analysis provides a high reassurance against cancer.

Summary Objectives Because current guidelines recognise high‐grade anal squamous intraepithelial lesions (HSILs) and low‐grade SILs (LSILs), and recommend treatment of all HSILs although not all progress to cancer, this study aims to distinguish transforming and productive HSILs by grading immunohistochemical (IHC) biomarkers p16INK4a (p16) and E4 in low‐risk human papillomavirus (lrHPV) and high‐risk (hr)HPV‐associated SILs as a potential basis for more selective treatment. Methods Immunostaining for p16 and HPV E4 was performed and graded in 183 biopsies from 108 HIV‐positive men who have sex with men. The causative HPV genotype of the worst lesion was identified using the HPV SPF10‐PCR‐DEIA‐LiPA25 version 1 system, with laser capture microdissection for multiple infections. The worst lesions were scored for p16 (0–4) to identify activity of the hrHPV E7 gene, and panHPV E4 (0–2) to mark HPV production and life cycle completion. Results There were 37 normal biopsies, 60 LSILs and 86 HSILs, with 85% of LSILs caused by lrHPV and 93% of HSILs by hrHPV. No normal biopsy showed E4, but 43% of LSILs and 37% of HSILs were E4 positive. No differences in E4 positivity rates were found between lrHPV and hrHPV lesions. Most of the lesions caused by lrHPV (90%) showed very extensive patchy p16 staining; p16 grade in HSILs was variable, with frequency of productive HPV infection dropping with increasing p16 grade. Conclusions Combined p16/E4 IHC identifies productive and nonproductive HSILs associated with hrHPV within the group of HSILs defined by the Lower Anogenital Squamous Terminology recommendations. This opens the possibility of investigating selective treatment of advanced transforming HSILs caused by hrHPV, and a ‘wait and see’ policy for productive HSILs. What's already known about this topic? For preventing anal cancer in high‐risk populations, all patients with high‐grade squamous intraepithelial lesions (HSILs) are treated, even though this group of lesions is heterogeneous, the histology is variable and regression is frequent. What does this study add? By adding human papillomavirus (HPV) E4 immunohistochemistry to p16 INK4a (p16), and grading expression of both markers, different biomarker expression patterns that reflect the heterogeneity of HSILs can be identified. Moreover, p16/E4 staining can separate high‐risk HPV‐associated HSILs into productive and more advanced transforming lesions, providing a potential basis for selective treatment.